The ultimate objective of the proposed research is to elucidate the mechanism and regulation of the facilitated transport of nucleosides, purines and phosphate in cultured animal cells and hepatocytes, to analyze the relationship between substrate transport and phosphorylation operating in tandem in the overall uptake process. We have developed rapid kinetic techniques which allow uptake measurements in time intervals as short as 1.5 sec, and steady-state equations which describe the relationship between substrate transport and subsequent phosphorylation. The methods allow the accurate determination of the kinetics of these processes operating in situ. With these methods we propose to further characterize the nucleoside, purine and phosphate transport systems of these cells with respect to substrate specificity, effects of sulfhydryl reactive agents, reducing and oxidizing agents, cAMP and the inhibition by nitrobenzylthioinosine which binds to the nucleoside carrier with a KD of about 1 nM. We will continue our estimation of the true association constant of the nucleoside carrier with its natural substrates and of the various resistance factors related to the "movement" of the carrier within the membrane. Further studies will deal with the relationship between nucleoside and purine transport and phosphorylation as a function of growth and the metabolic stability of the carrier. We will attempt to isolate transport mutants and to identify and characterize the nucleoside carrier in the membrane. This will involve transport studies with membrane vesicles, controlled extraction of proteins from the membranes, and binding studies and affinity chromatography with nitrobenzylthioinosine.